Preparation of anti-rh globulin from human plasma



June 10, 1969 w. PoLLAcK 3,449,314 PREPARATION OF ANT-.[*Rh GLOBULN FROM HUMAN PLASMA Filed'dune 3, 1966 United States Patent Office 3,449,314 Patented June 10, 1969 3,449,314 PREPARATION F ANTI-Rh GLBULIN FROM HUMAN PLASMA William Pollack, Belle Mead, NJ., assignor to Ortho Pharmaceutical Corporation, a corporation of New Jersey Filed .lune 3, 1966, Ser. No. 555,175 Int. Cl. A23j 1/06; C07g 7/00; A61k 23/ 00 U.S. Cl. 260--112 2 Claims The present invention relates to the production of gamma globulin and more particularly to a method of production of anti-Rh globulin.

Erythroblastosis fetalis is a herolytic disease which may cause brain damage, heart damage or death in the newborn child. This disease is caused by placenta-passing anti-bodies against fetal erythrocytes from a pregnant woman to her fetus. The disease is common in the children of parents whose blood have differing Rhfactors, as for example, the children of an Rh-uegative mother and an Rh-positive father.

When a woman whose Rh-factor differs from that of her husband becomes pregnant for the first time, red blood cells from the fetus may pass into her circulation. If the woman is Rh-negative and her husband is Rh-positive, the fetal red blood cells are usually Rhpositive. Antibodies to the Rh-positive red blood cell-s only rarely develop in the mothers circulation during the rst pregnancy and the first child is unaffected. Subsequent to the pregnancy, the `Rh-positive fetal erythrocytes remaining in the maternal circulation may immunize the mother so that she has about a 9 to 20% chance of producing Rh-antibodies. During subsequent pregnancies, the Rh-positive antibodies in the mothers `circulation pass through the placenta into the circulation of the fetus and destroy the fetal red blood cells causing erythroblastosis fetalis. Since about 15 percent of the population are Rh-negative, the disease is not uncommon.

Freda, Gorman and Pollack have reported that the intramuscular injection of anti-Rh gamma globulin into an Rh-negative mother within 72 hours of partruition will prevent the production in her circulation of antibodies which may cause erythroblastosis fetalis in the fetus of her next pregnancy (Science, Feb. 18, 1966, vol. 151, No. 3712, pages 823-830).

Cohn, U.S. Patent No. 2,390,074 discloses a method of fractionating blood by which gamma globulins are prepared. The gamma globulins prepared by the Cohn method contain 19 S globulin, plasminogen and lipids. While this gamma globulin is eminently suitable for prophylaxis against diseases such as measles and tetanus, the presence of the 19 S globulin, plasminogen and lipids are unnecessary contaminants and may decrease its effectiveness in preventing immunization to the Rhfactor on the fetal erythrocytes.

The anti-Rh globulin of the present invention is prepared from human plasma which contains albumin, plasminogen, alpha, beta and gamma globulins and various lipids. Specifically, the anti-Rh globulin of the invention is a gamma G globulin.

The fractionation of human plasma toy obtain anti-Rh globulin is carried out according to the accompanying flow sheet. The ability to fractionate human plasma is dependent upon the solubility of the various components of the plasma. At each stage of the fractionation, the separation of the fraction and the ultimate removal of those components which are undesirable in the anti-Rh globulin are determined by the critical control of pH, temperature, concentration of the precipitant and the ionic strength of the system.

Various organic solvents of low dielectric constant such as acetone and alcohols, precipitate proteins and have been used in the fractionation of plasma. The organic solvent utilized in the method of this invention is methanol. The ability to maintain the critical ionic vStrengths at the various stages of the fractionation is dependent upon the use of methanol.

In order to prevent denaturation of the proteins during fractionation, precipitation is carried out :at low temperatures. Since protein solubility is temperature dependent, the temperature chosen for each step of the fractionation must be the lowest possible which permits the desired separation in order to prevent denaturation.

Referring to the ilowsheet, the fractionation proceeds from whole human plasma. The plasma is cooled to about 1 C. and is then centrifuged to separate a cold insoluble precipitate from a supernatant. The supernatant is further fractionated to yield Precipitate I and Supernatant I. Precipitate I which consists principally of fibrinogen is discarded. Supernatant I is further fractionated to yield Supernatant IIA-III and Precipitate lI-l-III. Supernatant `II-l-[EIL which is discarded, contains alpha and beta globulin and lipids. Precipitate II-l-III consists principally of beta and gamma globulins and isoagglutinins, but also contains prothrombin, plasminogen, cholesterol and other lipids. Precipitate lI-l-III, upon further fractionation yields Supernatant II-l-III W and Precipitate II-i-III W. The beta globulin, cholesterol and other lipids are largely removed in Supernatant II-i-III W which is discarded. Precipitate II-l-III W consists principally of gamma globulins, isoagglutinins, plasminogen and prothrombin and some beta globulin, cholesterol and other lipids. Upon further fractionation, Precipitate II-l-III W yields Supernatant IIl-l-Precipitate III. Precipitate III, which is discarded, contains isoagglutinins, plasminogen and prothrombin, Supernatant III consists principally of gamma globulins and minor amouts of fibrinogen and lipids. The final step of the fractionation yields Precipitate II which is essentially pure gamma G globulin almost completely free of 19 S globulin, plasminogen and lipids. Precipitate II prepared by the process of the invention is an anti-Rh gamma globulin.

The invention may be more fully understood with reference to the following example.

Human blood plasma is cooled to 1il C. The plasma is centrifuged in a Sha-rples Super-Centrifuge with a 3-wing in bowl, at lil C. feeding at the ra-te of 1000i50 ml. per minute. The cold insoluble precipitate is discarded. The pH of the supernatant is adjusted to 7.2i0.2. The methanol content of the batch is brought to 10.7i0.l% (V./v.) by the addition of 177 ml. of 71% (v./v.) methanol per liter of plasma. The solution is stirred slowly at -5i0.5 C. for one hour and is maintained at -5i0.5 C. overnight. The solution is centrifuged in a Sharples Super-Centrifuge with a 3-wing in bowl at -5i0.5 C., feeding at the rate of 1000il00 ml. per minute. Supernatant I is collected in a bowl equipped with cooling device.

To each liter of Supernatant I there is added, with cooling, 601 ml. of 71% (v./v.) methanol, 0.88 ml. of 10 N acetic acid, and 0.44 ml. of 4 N sodium acetate. This brings the methanol concentration to 33.3%, the ionic strength to 0.086 gi./liter and the pH to 6.9. The solution is brought to a temperature of 5i0.5 C. and is stirred slowly for two hours. The solution is then allowed to stand at -5.5i0.5 C. for 16 hours.

The solution is centrifuged in a Sharples Super-Centrifuge with a 3-wing in bowl at -5l 0.5 C., at a rate of 1000 mLilOO rnl. per minute. Precipitate II-l-III is removed from the bowl, weighed, and is stored at a temperature of 20 C.

Maintaining a temperature of C., Precipitate II-l-III is suspended in 2 volumes (2 times original weight of Precipitate II-i-III) of 0 C. water in equilibrium with ice by cutting up the paste with a stainless steel spatula in a stainless steel pot and stirring until the suspension is uniform. 3 volumes (3 times original weight of Precipitate II-i-III) of 0.0187 M disodium phosphate solution is added to the suspension with stirring. 20 volumes (20 times original Weight of Precipitate II-i-III) of Water at 0 C. is added and the stirring at 0 C. is continued for 30-60 minutes. The pH is 7.2i0.2. To obtain a methanol percentage of 26.7i0.2%, 15 volumes (15 times the original Weight of Precipitate 114-111) of 71% (v./v.) methanol at C. is added. The ionic strength of the solution is 0.0052i0-0005 gi./liter.

The solution is brought to a temperature of 5 C. and is centrifuged in the Sharples Super-Centrifuge with a 3-wing in bowl at a temperature of 5.5 10.5 C. at a rate Of 500i50 ml. per minute. Precipitate II-i-III W is removed from the bowl and is weighed.

Precipitate II-l-III W is suspended in 2 volumes (2 times weight of Precipitate II-l-III W) of ice water. To the suspension is added 2 volumes (2 times weight of Precipitate II-l-III W) of 0.175 M sodium acetate. A further 1 volume (1 times weight of Precipitate II-i-III W) of ice water containing 0.216 ml. of acetate buffer for each gram of Precipitate II-l-III W paste. The acetate buffer is prepared by diluting 40 ml. of 10 N acetic acid and 20 ml. of 4 N sodium acetate to 100 ml. with distilled water. The pH of the solution is 5.2i0.2. The solution is stirred slowly for one hour. There is then added 13.5 volumes (13.5 times the original weight of Precipitate II-l-III W) of ice water and 8.66 volumes (8.66 times the original weight of Precipitate II|III W) of 71% (v /v.) methanol while maintaining the temperature atv 6i0.5 C. The solution is stirred slowly for one hour and is allowed to stand overnight. The solution contains 22.7% methanol and has a pH of 5.2 and an ionic strength of 0.015 gi./liter.

The solution is centrifuged in a Sharples Super-Centrifuge with a 3-wing in bowl, at a temperature of -6i0.5 C., and at a flow rate of 500i50 ml. per minute. The volume of Supernatant III is measured. Precipitate III is discarded.

Supernatant III is treated with 0.4% w./v. Celite 512 and is stirred for 20 minutes. The mixture is ltered through an Eitel lter press containing D-S pads.

An acetate buffer of pH 5.2 is made up as follows: 25 ml. 4 N sodium acetate and 3 ml. 10 N acetic acid made up to 100 ml. with distilled water. The acetate buer is diluted 100 times with 22.7% methanol. This solution has an ionic strength of 0.01 gi./liter. A-n Eitel filter press is precoated with the acetate buffer plus Celite 512 (0.5 gm./square inch of pad surface) using 40 ml. of acetatemethanol buffer/square inch of pad surface. Supernatant III is filtered and to the filtrate is added 50 millimoles (2.923 grams) of sodium chloride and 7.65 ml. of 1 M sodium bicarbonate per liter of filtered Supernatant III. The pH of the ltrate is 7.21 0.2.

To the ltrate is added 160 volumes of 100% methanol at a temperature below 5 C. The mixture is stirred slowly for 1 hour at a temperature of 6.5 i0.5 C. The mixture is permitted to stand overnight.

The solution is centrifuged in a Sharples Super-Centrifuge with 3-wing in bowl, at a temperature of -6i0.5 C. and at a ow rate of 500i50 ml. per minute. The supernatant is discarded.

Precipitate II is removed, suspended in 4 volumes of ice water and freeze-dried. The freeze-dried powder is weighed and is stored below at a temperature of 5 C.

Precipitate II as prepared 'by this example is 99% gamma G globulin free from plasminogen, lipids and 19 S globulins, having an electrophoretic mobility not greater than 1.1)(10-5 cm.2/volt/sec. in veronal buffer, pH 8.6, ionic strength 0.1 gi./liter. Precipitate II also contains about 0.99% sodium chloride.

What is claimed is:

1. A method of preparing an anti-Rh gamma globulin from human plasma which comprises the steps of:

(a) cooling human plasma to about 1 C. and separating from said plasma a rst supernatant and a first precipitate;

(b) adding methanol to said rst supernatant and adjusting the temperature to about 2 C., the amount of said methanol being between 10.5% and 10.9%, the pH being between 7 and 7.4 and the ionic strength being 0.136 gi./liter and separating from the resulting liquid system a second precipitate and a second supernatant;

(c) adding methanol to said second supernatant and adjusting the temperature to about 5 C., the amount of said methanol being between 33.25% and 33.35%, the pH being between 6.7 and 7.1 a-nd the ionic strength being between 0.081 and 0.091 gi./liter and separating from the resulting liquid system a third precipitate and a third supernatant;

(d) adding methanol to said third precipitate and adjusting the temperature to about 5 C., the amount of said methanol being between 26.5% and 26.9%, the pH being between 7.0 and 7.4 and the ionic strength being between 0.0047 and 0.0057 gi./liter and separating from the resulting liquid system a fourth precipitate and a fourth supernatant;

(e) adding methanol to said fourth precipitate and adjusting the temperature to about 6 C., the amount of said methanol being between 22.5% and 22.9%, the pH being between 5.0 and 5.4 and the ionic strength being between 0.008 and 0.018 gi./liter and separating from the resulting liquid system a fth precipitate and a fifth supernatant;

(f) adding methanol to said fifth supernatant and adjusting the temperature to about 6.5 C., the amount of said methanol being between 33.0% and 33.4%, the pH being between 7.0 and 7.4 and the ionic strength being between 0.055 and 0.065 gi./1iter and separating said resulting liquid system into a sixth precipitate and a sixth supernatant.

2. A method according to claim 1, wherein said sixth precipitate is freeze-dried.

References Cited Journal of Physiology, 1934, vol. VIII, pp. 97-109, Lui. Journal of Biological Chemistry, vol. 158, pp. 299-301.

WILLIAM H. SHORT, Primary Examiner.

HOWARD SHAIN, Assistant Examiner.

U.S. Cl. X.R. 424-11, 101 

1. A METHOD OF PREPARING AN ANTI-RH GAMMA GLOBULIN FROM HUMAN PLASMA WHICH CONPRISES THE STEPS OF: (A) COOLING HUMAN PLASMA TO ABOUT 1*C. AND SEPARATING FROM SAID PLASMA A FIRST SUPERNATANT AND A FIRST PRECIPITATE; (B) ADDING METHANOL TO SAID FIRST SUPERNATANT AND ADJUSTING THE TEMPERATURE TO ABOUT-2*C., THE AMOUNT OF SAID METHANOL BEING BETWEEN 10.5% AND 10.9%, THE PH BEING BETWEEN 7 AND 7.4 AND THE IONIC STRENGTH BEING 0.136 GI./LITER AND SEPARATING FROM THE RESULTING LIQUID SYSTEM A SECOND PRECIPITATE AND A SECOND SUPERNANT; (C) ADDING METHANOL TO SAID SECOND SUPERNATANT AND ADJUSTING THE TEMPERATURE TO ABOUT -5*C., THE AMOUNT OF SAID METHANOL BEING BETWEEN 33.25% AND 33.35%, THE PH BEING BETWEEN 6.7 AND 7.1 AND THE IONIC STRENGTH BEING BETWEEN 0.081 AND 0.091 GI./LITER AND SEPARATING FROM THE RESULTING LIQUID SYSTEM A THIRD PRECIPITATE AND A THIRD SUPERNATANT; (D) ADDING METHANOL TO SAID THIRD PRECIPITATE AND ADJUSTING THE TEMPERATURE TO ABOUT-5*C., THE AMOUNT OF SAID METHANOL BEING BETWEEN 26.5% AND 26.9%, THE PH BEING BETWEEN 7.0 AND 7.4 AND THE IONIC STRENGTH BEING BETWEEN 0.0047 AND 0.0057 GI./LITER AND SEPARATING FROM THE RESULTING LIQUID SYSTEM A FOURTH PRECIPITATE AND A FOURTH SUPERNATANT; (E) ADDING METHANOL TO SAID FOURTH PRECIPITATE AND ADJUSTING THE TEMPERATURE TO ABOUT-6*C., THE AMOUNT OF SAID METHANOL BEING BETWEEN 22.5% AND 22.9%, THE PH BEING BETWEEN 5.0 AND 5.4 AND THE IONIC STRENGTH BEING BETWEEN 0.008 AND 0.018 GI./LITER AND SEPARATING FROM THE RESULTING LIQUID SYSTEM A FIFTH PRECIPITATE AND A FIFTH SUPERNATANT; (F) ADDING METHANOL TO SAID FIFTH SUPERNATANT AND ADJUSTING THE TEMPERATURE OF ABOUT-6.5*C., THE AMOUNT OF SAID METHANOL BEING BETWEEN 33.0% AND 33.4%, THE PH BEING BETWEEN 7.0 AND 7.4 AND THE IONIC STRENGTH BEING BETWEEN 0.055 AND 0.065 GI./LITER AND SEPARATING SAID RESULTING LIQUID SYSTEM INTO A SIXTH PRECIPITATE AND A SIXTH SUPERNATANT. 